JP6848047B2 - Spiroindron polyethylene glycol carbonate-based compound and its composition, preparation method and its use - Google Patents

Description

The present invention belongs to the pharmaceutical field and relates to a spiroindolone polyethylene glycol carbonate-based compound and its composition, a method for producing the same, and its use as an anticancer agent due to its antitumor activity.

Cancer threatens human health and life. In recent years, research on anti-cancer agents has turned to research and development of specific molecular-targeted therapeutic agents.
Tumor-suppressing protein p53 plays an important role in preventing tumor development. In about 50% of human cancers, mutations or deletions in the gene encoding the p53 protein inactivate transcriptional activity and tumor suppressor function. For the remaining 50%, direct action between p53 and the human-mouse double microchromosome 2 (MDM2) protein plays an important role in inhibiting the function of wild-type p53. Interfering with the interaction between MDM2-p53 using small molecules is considered as a new cancer treatment strategy.

Since 2005, Wang Xiao Moe has reported a series of Spiro-Indolone analogs as inhibitors of the interaction between MDM2-p53, one of these series of compounds (SAR405838 / MI-77301). Has now entered the clinical development stage (see US7759383B2, US822228B2, US868132B2, US2013030173A1, WO20120652A2 and WO2012155066A2). The Swiss company F. Hoffmann La Roche also reported a series of spiro-indron analogs (see WO2011067185, WO2011134925 and WO201222707) and a series of pyrrolidine analogs (WO2013178570, WO20142068666 and WO201550000945), of which pyrrolidine analogs. Of these, RG7388 has entered the clinical development stage. Since these compounds have been confirmed to have limited solubility, the development of stable formulations faces great challenges in in vivo and clinical studies.

Conventional compounds are currently used only in oral preparations because they are inferior in water solubility, but because of their serious gastrointestinal effects, they are not highly bioavailable and affect the therapeutic effect of clinical tumors. Therefore, it is required to develop a spiro-indron analog having good water solubility, low toxicity, applicability to intravenous injection preparations, and higher activity as an inhibitor of MDM2-p53 interaction.

Therefore, an object of the present invention is a spiroindolone polyethylene glycol carbonate-based compound having good water solubility, low toxicity, applicability to intravenous injection preparations, and high activity, a stereoisomer thereof, or a pharmaceutically acceptable compound thereof. To provide salt.

Another object of the present invention is to provide a method for preparing the above-mentioned spiroindolone polyethylene glycol carbonate-based compound.
Another object of the present invention is to provide an antitumor pharmaceutical composition.
Another object of the present invention is to provide the use of the above-mentioned spiroindolone polyethylene glycol carbonate-based compounds, stereoisomers thereof or pharmaceutically acceptable salts thereof in the preparation of therapeutic agents for cancer.
Yet another object of the present invention is to provide a method of treating cancer.

（Ｉ）
一般式Ｉにおいて、
Ｙ_１、Ｙ_２、Ｙ_３及びＹ_４はそれぞれ独立してＨ及びハロゲンから選ばれ、
Ｒ_１はＨ及びＣ１−Ｃ５アルキル基から選ばれ、
Ｒ_２はＨ、Ｃ１−Ｃ５アルキル基及びＣ１−Ｃ５アルコキシ基から選ばれ、
Ｒ_３はＣ１−Ｃ１０アルキル基及びＣ２−Ｃ１０アルケニル基から選ばれ、
ｎは１〜８０の整数である。 According to one aspect of the present invention, the present invention provides a spiroindolone polyethylene glycol carbonate-based compound represented by the following general formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

(I)
In general formula I
Y ₁ , Y ₂ , Y ₃ and Y ₄ are independently selected from H and halogen, respectively.
R ₁ is selected from H and C1-C5 alkyl groups
R ₂ is selected from H, C1-C5 alkyl and C1-C5 alkoxy group,
R ₃ is selected from the C1-C10 alkyl group and the C2-C10 alkenyl group.
n is an integer from 1 to 80.

（ＩＩＩ）

（ＩＶ）
ここで、Ｙ_１、Ｙ_２、Ｙ_３、Ｙ_４、Ｒ_１、Ｒ_２、Ｒ_３及びｎは上記と同義である。 According to another aspect of the present invention, the method for producing the above-mentioned spiroindolone polyethylene glycol carbonate-based compound provided by the present invention includes the following spiroindolone-based compound represented by the general formula III and the following general. It comprises a step of subjecting a compound represented by the formula IV to a nucleophilic substitution reaction in the presence of a base to obtain the spiroindolone polyethylene glycol carbonate-based compound.

(III)

(IV)
Here, Y ₁ , Y ₂ , Y ₃ , Y ₄ , R ₁ , R ₂ , R ₃ and n are synonymous with the above.

According to another aspect of the present invention, the antitumor pharmaceutical composition provided by the present invention is a therapeutically effective amount of a spiroindolone polyethylene glycol carbonate-based compound represented by the above general formula I, a stereoisomer thereof or a stereoisomer thereof. It comprises the pharmaceutically acceptable salt and one or more pharmaceutically acceptable adjuvants.

According to yet another aspect of the present invention, the present invention is a spiroindolone polyethylene glycol carbonate-based compound represented by the general formula I in the preparation of a therapeutic agent for cancer, a stereoisomer thereof or a pharmaceutically acceptable salt thereof. Provide use of.

According to yet another aspect of the present invention, the present invention cancers a therapeutically effective amount of a spiroindolone polyethylene glycol carbonate-based compound represented by the general formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. Provided is a method of treating cancer to be administered to a patient.

The spiroindron polyethylene glycol carbonate compound of the present invention, its stereoisomer or a pharmaceutically acceptable salt thereof has excellent tumor inhibitory activity, has good water solubility, has low toxicity, and is injected intravenously. Can be prepared for use.

^{6 is a 1} H NMR spectrum diagram of the compound prepared in Example 6. ^{6 is a 1} H NMR spectrum diagram of the compound of the present invention prepared in Example 8. ^{FIG. 5 is a 1} H NMR spectrum diagram of the compound prepared in Example 10. ^{FIG. 5 is a 1} H NMR spectrum diagram of the compound of the present invention prepared in Example 11. FIG. 5 is a spectrum diagram by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of the compound of the present invention prepared in Example 13. FIG. 5 is a spectrum diagram by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) of the compound of the present invention prepared in Example 14. Adriamycin, racem-SIP, racem-SIP-PEG-2 (ie, compound of Example 8), racem-SIP-PEG-3 (ie, compound of Example 9), chiral-R-SIP-PEG-3 (ie, compound of Example 9) That is, SJSA-1 nude mouse transplanted tumors of (the compound of Example 11), racem-SIP-PEG-7 (ie, the compound of Example 12) and racem-SIP-PEG-2000 (ie, the compound of Example 14). It is a graph of the relative tumor volume which shows the inhibitory effect on the growth of. Adriamycin, racem-SIP, racem-SIP-PEG-2 (ie, compound of Example 8), racem-SIP-PEG-3 (ie, compound of Example 9), chiral-R-SIP-PEG-3 (ie, compound of Example 9) That is, SJSA-1 nude mouse transplanted tumors of (the compound of Example 11), racem-SIP-PEG-7 (ie, the compound of Example 12) and racem-SIP-PEG-2000 (ie, the compound of Example 14). It is a figure which shows the inhibitory effect on the growth of. Adriamycin, racem-SIP, racem-SIP-PEG-2 (ie, compound of Example 8), racem-SIP-PEG-3 (ie, compound of Example 9), chiral-R-SIP-PEG-3 (ie, compound of Example 9) That is, the weight of SJSA-1 nude mice of Example 11 compound), racem-SIP-PEG-7 (ie, compound of Example 12) and racem-SIP-PEG-2000 (ie, compound of Example 14). It is a graph which shows the influence on.

（ＩＩ）
ここで、Ｙ_１、Ｙ_２、Ｙ_３、Ｙ_４、Ｒ_１、Ｒ_２及びｎは上記と同義である。 Hereinafter, the present invention will be specifically described.
According to the spiroindolone polyethylene glycol carbonate-based compound of the present invention, its steric isomer or a pharmaceutically acceptable salt thereof, in a preferred embodiment, the spiroindolone polyethylene glycol carbonate-based compound is described in the following general formula II. It is preferably a spiroindolone polyethylene glycol carbonate-based compound represented by.

(II)
Here, Y ₁ , Y ₂ , Y ₃ , Y ₄ , R ₁ , R ₂ and n are synonymous with the above.

In a more preferred embodiment
It is preferable that Y ₁ , Y ₂ , Y ₃ and Y ₄ are independently selected from H, F and Cl, respectively.
R ₁ is preferably selected from H and C1-C3 alkyl groups, more preferably a methyl group or an ethyl group, and most preferably a methyl group.
R ₂ is preferably selected from C1-C5 alkoxy group, and more preferably a methoxy group or an ethoxy group, most preferably a methoxy group.
R ₃ is preferably selected from the C1-C6 alkyl group, more preferably a pentyl group, and most preferably a neopentyl group.
n is preferably an integer of 1 to 60, more preferably an integer of 2 to 50, and most preferably an integer of 3 to 50.

The spiroindolone polyethylene glycol carbonate-based compound represented by the general formula I according to the present invention can exhibit tautomerism or structural isomerism. This is because the present invention comprises any tautomeric or structural isomer of these compounds, or a mixture thereof, and any tautomer or structural isomer represented by the above structural formula. It shows that it is not limited to.

The pharmaceutically acceptable salt of the spiroindron polyethylene glycol carbonate-based compound according to the present invention is a general acid addition salt composed of the compound and an appropriate non-toxic organic acid or non-toxic inorganic acid, or the said. It means a general base addition salt consisting of a compound and a suitable non-toxic organic base or non-toxic inorganic base. Examples of the inorganic acid include hydrochloric acid, sulfuric acid, phosphoric acid, nitrate and the like, and examples of the organic acid include p-toluenesulfonic acid, salicylic acid, oxalic acid, citric acid, lactic acid, malic acid and the like, and base addition salts. Examples include ammonium salts, sodium salts, potassium salts and quaternary ammonium hydroxides.

（ＩＩＩ）

（ＩＶ）
ここで、Ｙ_１、Ｙ_２、Ｙ_３、Ｙ_４、Ｒ_１、Ｒ_２、Ｒ_３及びｎは上記と同義である。
ここで、一般式ＩＩＩで表されるスピロインドロン系化合物は、ＷＯ２０１１／０６７１８５Ａ１及びＷＯ２０１１／１３４９２５Ａ１に記載の方法を参照して調製することができる。
一般式ＩＶで表される化合物は、例えば以下の反応により調製することができる。

In the method for producing a spiroindolone polyethylene glycol carbonate-based compound provided by the present invention, a spiroindolone-based compound represented by the following general formula III and a compound represented by the following general formula IV are based. In the presence, it comprises a step of subjecting to a nucleophilic substitution reaction to obtain the spiroindron polyethylene glycol carbonate-based compound.

(III)

(IV)
Here, Y ₁ , Y ₂ , Y ₃ , Y ₄ , R ₁ , R ₂ , R ₃ and n are synonymous with the above.
Here, the spiroindolone-based compound represented by the general formula III can be prepared by referring to the methods described in WO2011 / 067185A1 and WO2011 / 134925A1.
The compound represented by the general formula IV can be prepared, for example, by the following reaction.

The antitumor pharmaceutical composition provided by the present invention is a therapeutically effective amount of a spiroindolone polyethylene glycol carbonate-based compound represented by the above general formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof and a pharmacy. Includes a pharmaceutically acceptable adjuvant.

The antitumor pharmaceutical composition of the present invention can be injectable solution, tablets, capsules and other dosage forms, if necessary. The pharmaceutically acceptable adjuvant can be appropriately selected from common adjuvants depending on the dosage form prepared from the antitumor pharmaceutical composition. For example, in the case of a lyophilized product for injection, the pharmaceutically acceptable adjuvant may include excipients, diluents and the like.

INDUSTRIAL APPLICABILITY The present invention provides the use of a spiroindolone polyethylene glycol carbonate-based compound represented by the general formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in the preparation of a therapeutic agent for cancer.

According to the present invention, a method for treating cancer in which a therapeutically effective amount of a spiroindron polyethylene glycol carbonate-based compound represented by the general formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is administered to a cancer patient. provide.

The cancers include bladder cancer, breast cancer, colon cancer, rectal cancer, renal cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, bile sac cancer, ovarian cancer, pancreatic adenocarcinoma, gastric cancer, cervical cancer, and thyroid cancer. , Prostate cancer, skin cancer, acute lymphocytic leukemia, chronic osteoporosis leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-hodgkin's lymphoma, hair cell lymphoma, Berkit It includes, but is not limited to, lymphoma, acute osteoporosis, melanoma, endometrial cancer, head and neck cancer, glioblastoma or osteosarcoma.

The compounds of the present invention may be administered by any suitable conventional method, which comprises oral administration, intravenous injection and local injection. The therapeutically effective amount of a compound of the present invention means an amount effective for preventing, alleviating or ameliorating the symptoms of a disease or prolonging the survival time of a patient.

The therapeutically effective amounts or doses of the compounds of the invention are variable in a wide range and are determined by methods known in the art. The above doses are tailored to the individual in each particular situation, including the specific compound administered, the route of administration, the disease being treated and the patient being treated. Usually, when orally or parenterally administered to an adult weighing about 70 kg, a daily dose of about 10 mg to about 10000 mg, preferably about 200 mg to about 1000 mg is appropriate, but if otherwise specified, the above upper limit is exceeded. It should be understood that it is okay. The daily dose is administered as a single dose or in divided doses, or as a continuous infusion in the case of parenteral administration.

Hereinafter, the present invention will be specifically described with reference to Examples, but the scope of the present invention is not limited to these Examples.

０℃で３−メトキシ安息香酸メチル（８３．０ｇ、５００ｍｍｏｌ）のＨ_２ＳＯ_４（７０ｗｔ％、２００ｍｌ）溶液にＨＮＯ_３（６５ｗｔ％、４０ｍｌ）を滴下し、得られた混合物を終夜撹拌し、その後、氷水に注入した。このようにして得られた混合物を濾過し、得られた固形ケーキを水で洗浄し（３×３００ｍｌ）、黄色の固体として４−ニトロ−３−メトキシ安息香酸メチル（８４．４ｇ、収率８０％）を得た。
４−ニトロ−３−メトキシ安息香酸メチル（８４．４ｇ、４００ｍｍｏｌ）のエタノール（１５００ｍｌ）溶液を、Ｐｄ／炭素（１０％Ｐｄ、５．３５ｇ）触媒の存在下で、Ｈ_２雰囲気中で５時間撹拌し、その後、この反応液をセライト^(R)珪藻土により濾過して触媒を除去し、溶剤を真空下で蒸発させ、灰白色の固体を得て、この灰白色の固体をメタノールで再結晶し、４−アミノ−３−メトキシ安息香酸メチル（７０．９５ｇ、３９２ｍｍｏｌ、収率９８％）を得た。
^１Ｈ ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３）：δ７．５５（ｄｄ，Ｊ＝８．２Ｈｚ， １．４Ｈｚ，１Ｈ），７．４５（ｄ，Ｊ＝８．０Ｈｚ，１Ｈ），６．６５（ｄ，Ｊ＝８．０Ｈｚ，１Ｈ），４．２２（ｓ，２Ｈ），３．９０（ｓ，３Ｈ），３．８６（ｓ，３Ｈ）；
ＭＳ：Ｃ_９Ｈ_１２ＮＯ_３（［Ｍ＋Ｈ］^＋）計算値：１８２；実測値：１８２。 Example 1: Synthesis of intermediate 4-amino-3-methoxymethyl benzoate

_{HNO 3} (65 wt%, 40 ml) was added dropwise to a solution of methyl 3-methoxybenzoate (83.0 g, 500 mmol) in H ₂ SO ₄ (70 wt%, 200 ml) at 0 ° C., and the resulting mixture was stirred overnight. Then, it was injected into ice water. The mixture thus obtained was filtered and the resulting solid cake was washed with water (3 x 300 ml) and as a yellow solid methyl 4-nitro-3-methoxybenzoate (84.4 g, yield 80). %) Was obtained.
4-nitro-3-methoxybenzoate (84.4 g, 400 mmol) in ethanol (1500 ml) solution of, Pd / carbon (10% Pd, 5.35g) in the presence of a catalyst, 5 hours in _{a H 2} atmosphere After stirring, the reaction ^{was filtered through Celite (R)} diatomaceous soil to remove the catalyst, the solvent was evaporated under vacuum to give a grayish white solid, and the grayish white solid was recrystallized from methanol and 4 -Methyl amino-3-methoxybenzoate (70.95 g, 392 mmol, yield 98%) was obtained.
^{1 1} H NMR (400 MHz, CDCl ₃ ): δ7.55 (dd, J = 8.2 Hz, 1.4 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 8.0Hz, 1H), 4.22 (s, 2H), 3.90 (s, 3H), 3.86 (s, 3H);
MS: C ₉ H ₁₂ NO ₃ ([M + H] ⁺ ) Calculated value: 182; Measured value: 182.

Ｎ_２雰囲気中で、２−（（ｔ−ブトキシカルボニル）アミノ）酢酸（８．７５ｇ，５０ｍｍｏｌ）、Ｅｔ_３Ｎ（６０ｍｍｏｌ）の塩化メチレン（ＤＣＭ）（１５０ｍｌ）と塩化アセチル（６０ｍｍｏｌ）の溶液を室温で１時間攪拌し、その後、４−アミノ−３−メトキシ安息香酸メチル（９．０５ｇ、５０ｍｍｏｌ）のエタノール（１００ｍｌ）溶液を添加し、得られた混合物を終夜攪拌した。次いで、反応液に２００ｍｌの水を添加し、この反応混合物をＤＣＭで抽出し（２×１５０ｍｌ）、有機層を合わせ、そして、水で洗浄し、Ｎａ_２ＳＯ_４により乾燥した後、溶剤を真空下で除去し、得られた残留物をシリカゲルカラムクロマトグラフィーにより精製し、白色の固体として目的化合物（１２．０ｇ、収率７０％）を得た。
^１Ｈ ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３）：δ８．５９（ｓ，１Ｈ），８．４２（ｄ， Ｊ＝８．４Ｈｚ，１Ｈ），７．６６（ｄ，Ｊ＝８．４Ｈｚ，１Ｈ），７．５２（ｓ，１ Ｈ），５．３０（ｓ，１Ｈ），３．９６（ｄ，Ｊ＝５．２Ｈｚ，２Ｈ），３．９１（ｓ， ３Ｈ），３．８９（ｓ，３Ｈ），１．４８（ｓ，９Ｈ）；
ＭＳ：Ｃ_１６Ｈ_２３Ｎ_２Ｏ_６（［Ｍ＋Ｈ］^＋）計算値：３３９；実測値：３３９．２。 Example 2: Synthesis of intermediate 4- (2-((t-butoxycarbonyl) amino) acetylamino) -3-methoxymethyl benzoate

In an N ₂ atmosphere, 2 - ((t- butoxycarbonyl) amino) acetate (8.75 g, 50 _mmol), a solution of methylene chloride (DCM) (150 ml) and acetyl chloride (60 mmol) of Et 3 N (60 mmol) The mixture was stirred at room temperature for 1 hour, then a solution of methyl 4-amino-3-methoxybenzoate (9.05 g, 50 mmol) in ethanol (100 ml) was added and the resulting mixture was stirred overnight. The reaction mixture was then added with 200 ml of water, the reaction mixture was extracted with DCM (2 x 150 ml), the organic layers were combined and washed with water, dried with Na ₂ SO ₄ , and then the solvent was vacuumed. The residue was removed below and the obtained residue was purified by silica gel column chromatography to obtain the target compound (12.0 g, 70% yield) as a white solid.
^{1 1} H NMR (400 MHz, CDCl ₃ ): δ8.59 (s, 1H), 8.42 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7 .52 (s, 1H), 5.30 (s, 1H), 3.96 (d, J = 5.2Hz, 2H), 3.91 (s, 3H), 3.89 (s, 3H) , 1.48 (s, 9H);
MS: C ₁₆ H ₂₃ N ₂ O ₆ ([M + H] ⁺ ) Calculated value: 339; Measured value: 339.2.

４−（２−（（ｔ‐ブトキシカルボニル）アミノ）アセチルアミノ）−３−メトキシ安息香酸メチル（１１．８３ｇ、３５ ｍｍｏｌ）と濃塩酸（７．０ｍｌ）の酢酸エチル（１００ｍｌ）溶液を室温で終夜攪拌し、その後、この反応液を濾過し、得られた濾過ケーキを酢酸エチル（２×５０ｍｌ）で洗浄してから、乾燥し、白色の固体として４−（２−アミノアセチルアミノ）−３−メトキシ安息香酸メチル塩酸塩（６．５６２ｇ、収率６９％）を得た。
^１Ｈ ＮＭＲ（４００ＭＨｚ，ＤＭＳＯ）：δ１０．０３（ｓ，１Ｈ），８．４９（ｓ， ３Ｈ），８．２０（ｄ，Ｊ＝８．４Ｈｚ，１Ｈ），７．５７（ｄ，Ｊ＝８．４Ｈｚ，１ Ｈ），７．５１（ｄ，Ｊ＝１．２Ｈｚ，１Ｈ），３．９０（ｓ，５Ｈ），３．８３（ｓ， ３Ｈ）；
ＭＳ：Ｃ_１１Ｈ_１５Ｎ_２Ｏ_４（［Ｍ−ＨＣｌ＋Ｈ］^＋）計算値：２３９；実測値：２３９。 Example 3: Synthesis of Intermediate 4- (2-Aminoacetylamino) -3-methoxymethyl benzoate hydrochloride

A solution of 4- (2-((t-butoxycarbonyl) amino) acetylamino) -3-methoxymethyl benzoate (11.83 g, 35 mmol) and concentrated hydrochloric acid (7.0 ml) in ethyl acetate (100 ml) at room temperature. Stir overnight, then filter the reaction, wash the resulting filtered cake with ethyl acetate (2 x 50 ml), then dry and as a white solid 4- (2-aminoacetylamino) -3. -Methyl benzoate hydrochloride (6.562 g, yield 69%) was obtained.
^{1 1} H NMR (400 MHz, DMSO): δ10.03 (s, 1H), 8.49 (s, 3H), 8.20 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.4Hz, 1H), 7.51 (d, J = 1.2Hz, 1H), 3.90 (s, 5H), 3.83 (s, 3H);
MS: C ₁₁ H ₁₅ N ₂ O ₄ ([M-HCl + H] ⁺ ) Calculated value: 239; Measured value: 239.

室温で４−（２−アミノアセチルアミノ）−３−メトキシ安息香酸メチル塩酸塩（６．５６ｇ、２４ｍｍｏｌ）のＭＴＢＥ（メチル−ｔ−ブチルエーテル）（１００ｍｌ）懸濁液にトリエチルアミン（５ｍｌ）を添加し、１時間攪拌した後、３，３−ジメチル−ｎ−ブチルアルデヒド（２．６４ｇ、２６．４ｍｍｏｌ）を滴下し、得られた混合物を室温で１０時間攪拌した。その後、トリエチルアミン塩酸塩を除去するようにこの反応混合液を濾過し、そしてこの固形濾過ケーキをＭＴＢＥで洗浄し（３×３０ｍｌ）、有機相を合わせ、有機溶剤を真空下で蒸発させ、粘調な油状物を得て、精製せずにそのまま次のステップに使用した。 Example 4: Synthesis of intermediate (E) -4- (2-((3,3-dimethylbutylidene) amino) acetylamino) -3-methoxymethyl benzoate

Triethylamine (5 ml) was added to a MTBE (methyl-t-butyl ether) (100 ml) suspension of 4- (2-aminoacetylamino) -3-methoxymethyl benzoate (6.56 g, 24 mmol) at room temperature. After stirring for 1 hour, 3,3-dimethyl-n-butylaldehyde (2.64 g, 26.4 mmol) was added dropwise, and the obtained mixture was stirred at room temperature for 10 hours. The reaction mixture is then filtered to remove triethylamine hydrochloride, and the solid filtered cake is washed with MTBE (3 x 30 ml), the organic phases are combined, the organic solvent is evaporated under vacuum and viscous. Oil was obtained and used as is in the next step without purification.

ピペリジン（１ｍｌ）の存在下で、６−クロロジヒドロインドール−２−オン（８．３５ｇ、５０ｍｍｏｌ）及び３−クロロ−２−フルオロベンズアルデヒド（８．２９５ｇ、５２．５ｍｍｏｌ）を還流下で６時間撹拌し、その後、反応懸濁液を濾過し、得られた固体を収集してメタノールで洗浄し、そして乾燥し、生成物として黄色の固体の（Ｅ）−６−クロロ−３−（３−クロロ−２−フルオロベンジリデン）インドール−２−オン（１４．６３ｇ、４７．５ｍｍｏｌ、収率９５％）を得た。
^１Ｈ ＮＭＲ（４００ＭＨｚ，ＤＭＳＯ）：δ１０．８５（ｓ，１Ｈ），７．７０（ｑ， Ｊ＝７．３Ｈｚ，２Ｈ），７．５４（ｓ，１Ｈ），７．３６（ｔ，Ｊ＝８Ｈｚ，１Ｈ），７．１６（ｄ，Ｊ＝８Ｈｚ，１Ｈ），６．９２−６．８６（ｍ，２Ｈ）；
ＭＳ：Ｃ_１５Ｈ_９Ｃ_ｌ２ＦＮＯ（［Ｍ＋Ｈ］^＋）計算値：３０８；実測値：３０８．０。 Example 5: Synthesis of intermediate (E) -6-chloro-3- (3-chloro-2-fluorobenzylidene) dihydroindole-2-one

In the presence of piperidine (1 ml), 6-chlorodihydroindole-2-one (8.35 g, 50 mmol) and 3-chloro-2-fluorobenzaldehyde (8.295 g, 52.5 mmol) are stirred under reflux for 6 hours. The reaction suspension is then filtered, the resulting solid is collected, washed with methanol and dried, and the product is a yellow solid (E) -6-chloro-3- (3-chloro). -2-Fluorobenzylidene) indole-2-one (14.63 g, 47.5 mmol, 95% yield) was obtained.
^{1 1} H NMR (400 MHz, DMSO): δ10.85 (s, 1H), 7.70 (q, J = 7.3 Hz, 2H), 7.54 (s, 1H), 7.36 (t, J = 8Hz, 1H), 7.16 (d, J = 8Hz, 1H), 6.92-6.86 (m, 2H);
MS: C ₁₅ H _{9 Cl2} FNO ([M + H] ⁺ ) Calculated value: 308; Measured value: 308.0.

１，８−ジアザビシクロウンデカ−７−エン（７ｍｌ）の存在下で、（Ｅ）−４−（２−（（３，３−ジメチルブチリデン）アミノ）アセチルアミノ）−３−メトキシ安息香酸メチル（２４ｍｍｏｌ）と（Ｅ）−６−クロロ−３−（３−クロロ−２−フルオロベンジリデン）ジヒドロインドール−２−オン（２４ｍｍｏｌ）のトルエン（ｔｏｌｕｅｎｅ）（６０ｍｌ）溶液を終夜攪拌し、その後、１００ｍｌの水を添加し、得られた混合物を酢酸エチルで抽出し（２×１５０ｍｌ）、有機層を合わせ、そして、この有機層水で洗浄し、さらにＮａ_２ＳＯ４により乾燥し、溶剤を真空下で除去し、得られた残留物をシリカゲルカラムクロマトグラフィーにより精製し、黄色の固体として目的化合物（１３．９ｇ、収率９２％）を得た。
^１Ｈ ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３）：δ１０．６５（ｓ，１Ｈ），８．４９ （ｄ，Ｊ＝８Ｈｚ，１Ｈ），７．９４（ｓ，１Ｈ），７．６６（ｄｄ，Ｊ＝８．６Ｈｚ， Ｊ＝１Ｈｚ，１Ｈ），７．５７（ｓ，１Ｈ），７．５１（ｔ，Ｊ＝６．８Ｈｚ，１Ｈ）， ７．２８（ｓ，１Ｈ），７．１６（ｔ，Ｊ＝７．４Ｈｚ，１Ｈ），７．０７（ｄｄ，Ｊ ＝８．２Ｈｚ，Ｊ＝１．４Ｈｚ，１Ｈ），６．９２（ｔ，Ｊ＝８Ｈｚ，１Ｈ），６．７３（ｄ，Ｊ＝１．２Ｈｚ，１Ｈ），４．６９（ｔ，Ｊ＝８．８Ｈｚ，１Ｈ），４．４４ （ｄ，Ｊ＝９．２Ｈｚ，１Ｈ），３．９３（ｓ，３Ｈ），３．９０（ｓ，３Ｈ），３．６５（ｔ，Ｊ＝１０．４Ｈｚ，１Ｈ），３．２９（ｔ，Ｊ＝１０．８Ｈｚ，１Ｈ），１．３８−１．２８（ｍ，１Ｈ），０．９６（ｓ，９Ｈ），０．９２（ｓ，１Ｈ）（図１を参照）；
ＭＳ：Ｃ_３２Ｈ_３３Ｃｌ_２ＦＮ_３Ｏ_５（［Ｍ＋Ｈ］^＋）計算値：６２８；実測値：６２８．２。 Example 6: Intermediate Lasemi-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'- Synthesis of methyl neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxymethyl benzoate

In the presence of 1,8-diazabicycloundec-7-ene (7 ml), (E) -4-(2-((3,3-dimethylbutylidene) amino) acetylamino) -3-methoxy benzo A solution of methyl (24 mmol) acid (24 mmol) and (E) -6-chloro-3- (3-chloro-2-fluorobenzylidene) dihydroindole-2-one (24 mmol) in toluene (60 ml) was stirred overnight, followed by , 100 ml of water was added, the resulting mixture was extracted with ethyl acetate (2 x 150 ml), the organic layers were combined and washed with this organic layer water, further _{dried with Na 2} SO4 and the solvent vacuumed. The residue was removed below and the resulting residue was purified by silica gel column chromatography to give the desired compound (13.9 g, 92% yield) as a yellow solid.
¹ H NMR (400 MHz, CDCl ₃ ): δ10.65 (s, 1H), 8.49 (d, J = 8 Hz, 1H), 7.94 (s, 1H), 7.66 (dd, J = 8) .6Hz, J = 1Hz, 1H), 7.57 (s, 1H), 7.51 (t, J = 6.8Hz, 1H), 7.28 (s, 1H), 7.16 (t, J) = 7.4Hz, 1H), 7.07 (dd, J = 8.2Hz, J = 1.4Hz, 1H), 6.92 (t, J = 8Hz, 1H), 6.73 (d, J = 1.2Hz, 1H), 4.69 (t, J = 8.8Hz, 1H), 4.44 (d, J = 9.2Hz, 1H), 3.93 (s, 3H), 3.90 ( s, 3H), 3.65 (t, J = 10.4Hz, 1H), 3.29 (t, J = 10.8Hz, 1H), 1.38-1.28 (m, 1H), 0. 96 (s, 9H), 0.92 (s, 1H) (see FIG. 1);
MS: C ₃₂ H ₃₃ Cl ₂ FN ₃ O ₅ ([M + H] ⁺ ) Calculated value: 628; Measured value: 628.2.

２−（２−メトキシエトキシ）エタノール（６．０ｇ、５０ｍｍｏｌ）と１−クロロエチルカルボノクロリダート（１−ｃｈｌｏｒｏｅｔｈｙｌ ｃａｒｂｏｎｏｃｈｌｏｒｉｄａｔｅ）（７．４５５ｇ、５２．５ｍｍｏｌ）の溶液を、Ｅｔ_３Ｎ（４．０４ｇ、５２．５ｍｍｏｌ）の存在下で、室温で６時間攪拌し、その後、そこに２００ｍｌの水を添加し、この反応混合物をＤＣＭで抽出し（２×１５０ｍｌ）、有機層を合わせ、そして、水で洗浄し、さらにＮａ_２ＳＯ_４により乾燥し、溶剤を真空下で除去し、得られた残留物をシリカゲルカラムクロマトグラフィーにより精製し、油状の目的化合物（８．４７５ｇ、収率７５％）を得た。
^１Ｈ ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３）：δ６．４１（ｑ，Ｊ＝５．７Ｈｚ，１ Ｈ），４．３７−４．３２（ｍ，２Ｈ），３．７３（ｔ，Ｊ＝４．８Ｈｚ，２Ｈ），３．６６−３．６２（ｍ，２Ｈ），３．５６−３．５２（ｍ，２Ｈ），３．３７（ｓ，３Ｈ）， １．８１（ｄ，Ｊ＝６．４Ｈｚ，３Ｈ）。 Example 7: Synthesis of Intermediate 1-Chloroethyl (2- (2-methoxyethoxy) Ethyl) Carbonate

A solution of 2- (2-methoxyethoxy) ethanol (6.0 g, 50 mmol) and 1-chloroethyl carbonochlorodate (7.455 g, 52.5 mmol) was added to Et ₃ N (4. In the presence of (04 g, 52.5 mmol), the mixture was stirred at room temperature for 6 hours, after which 200 ml of water was added, the reaction mixture was extracted with DCM (2 x 150 ml), the organic layers were combined and then The mixture was washed with water, further _{dried with Na 2} SO ₄ , the solvent was removed under vacuum, and the obtained residue was purified by silica gel column chromatography to obtain an oily target compound (8.475 g, yield 75%). Got
^{1 1} H NMR (400 MHz, CDCl ₃ ): δ6.41 (q, J = 5.7 Hz, 1 H), 4.37-4.32 (m, 2H), 3.73 (t, J = 4.8 Hz) , 2H), 3.66-3.62 (m, 2H), 3.56-3.52 (m, 2H), 3.37 (s, 3H), 1.81 (d, J = 6.4Hz) , 3H).

ラセミ−４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキサスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸メチル（１３．８１６ｇ、２２ｍｍｏｌ）のＴＨＦ（２０ｍｌ）溶液にＬｉＯＨ（５．８ｍｌ、Ｈ_２Ｏ中５０％濃度）を添加し、終夜攪拌し、その後、反応液を塩酸でｐＨ＝１に酸性化し、そして得られた反応混合物を酢酸エチルで抽出し（３×５０ｍｌ）、有機層を合わせ、水で洗浄し、さらに無水Ｎａ_２ＳＯ_４により乾燥し、溶剤を真空下で除去し、得られた残留物をシリカゲルカラムクロマトグラフィーにより精製し、黄色の固体として対応する酸化合物（ラセミ−４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキソスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸）（１２．４３ｇ、収率９２％）を得た。
ラセミ−４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキソスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸（３０６ｍｇ、０．５ｍｍｏｌ）と１−クロロエチル（２−（２−メトキシエトキシ）エチル）カーボネート（５１０ｍｇ、２．２５ｍｍｏｌ）のジメチルホルムアミド（１０ｍｌ）溶液にＮａＯＨ（３ｍｍｏｌ、９７５ｍｇ、６ｅｑ）を添加し、窒素ガス雰囲気中で終夜攪拌し、その後、１０ｍｌの水を添加し、この混合物を酢酸エチルで抽出し （２×１５ｍｌ）、有機層を合わせ、水で洗浄し、さらに無水Ｎａ_２ＳＯ_４により乾燥し、溶剤を真空下で除去し、得られた残留物をシリカゲルカラムクロマトグラフィーにより精製し、黄色の固体として目的化合物（１０２ｍｇ、収率２６％）を得た。
^１Ｈ ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３）：δ１０．６２（ｓ，１Ｈ），８．４８ （ｄ，Ｊ＝８Ｈｚ，１Ｈ），７．７９（ｄ，Ｊ＝１．６Ｈｚ，１Ｈ），７．６６（ｄｄ， Ｊ＝８．６Ｈｚ，１．４Ｈｚ，１Ｈ），７．５６（ｓ，１Ｈ），７．４７（ｔ，１Ｈ），７．３３（ｄ，Ｊ＝８Ｈｚ，１Ｈ），７．２３（ｄ，Ｊ＝９．２Ｈｚ，１Ｈ），７．１９（ｄ，Ｊ＝７．６Ｈｚ，１Ｈ），７．０７−６．９８（ｍ，２Ｈ），４．７５−４．６８（ｍ，１Ｈ），４．５０−４．４２（ｍ，２Ｈ），４．３７−４．２５（ｍ，２Ｈ）， ３．９２（ｓ，３Ｈ），３．８２（ｔ，２Ｈ），３．７６−３．５０（ｍ，９Ｈ），２．１８（ｂｓ，１Ｈ），１．６５（ｄ，Ｊ＝５．２Ｈｚ，３Ｈ），１．３６−１．２２（ｍ，１Ｈ），０．９５（ｓ，９Ｈ）（図２を参照）。
ＭＳ：Ｃ_３９Ｈ_４５Ｃｌ_２ＦＮ_３Ｏ_１０（［Ｍ＋Ｈ］^＋）計算値：８０４；実測値：８０４．２ 。 Example 8: Racemic-9-oxo-2,5,8,10-tetraoxadodecane-11-yl-4-((2'S, 3R, 4'S, 5'R) -6-chloro-4 '-(3-Chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidine] -5'-carboxamide) -3-methoxybenzoic acid ester (racemic-SIP-) Synthesis of PEG-2)

Racem-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxaspiro [ dihydroindole-3,3'-pyrrolidin] -5'-carboxamido) -3-methoxybenzoate (13.816g, THF (20ml) was added LiOH (5.8ml, _{H 2} O in 50% strength 22 mmol) ) Is added and stirred overnight, after which the reaction solution is acidified to pH = 1 with hydrochloric acid, and the resulting reaction mixture is extracted with ethyl acetate (3 x 50 ml), the organic layers are combined and washed with water. , Further _{dried with anhydrous Na 2} SO ₄ , the solvent was removed under vacuum and the resulting residue was purified by silica gel column chromatography to give the corresponding acid compound as a yellow solid (Racemi-4-((2'). S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'- Pyrrolidine] -5'-carboxamide) -3-methoxybenzoic acid) (12.43 g, yield 92%) was obtained.
Lacem-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [ Dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoic acid (306 mg, 0.5 mmol) and 1-chloroethyl (2- (2-methoxyethoxy) ethyl) carbonate (510 mg, 2. NaOH (3 mmol, 975 mg, 6 eq) was added to a 25 mmol) dimethylformamide (10 ml) solution, stirred overnight in a nitrogen gas atmosphere, then 10 ml of water was added and the mixture was extracted with ethyl acetate (2). × 15 ml), the organic layers were combined, washed with water, further dried with anhydrous Na ₂ SO ₄ , the solvent was removed under vacuum and the resulting residue was purified by silica gel column chromatography to give a yellow solid. The target compound (102 mg, yield 26%) was obtained.
^{1 1} H NMR (400 MHz, CDCl ₃ ): δ10.62 (s, 1H), 8.48 (d, J = 8 Hz, 1H), 7.79 (d, J = 1.6 Hz, 1H), 7.66 (Dd, J = 8.6Hz, 1.4Hz, 1H), 7.56 (s, 1H), 7.47 (t, 1H), 7.33 (d, J = 8Hz, 1H), 7.23 (D, J = 9.2Hz, 1H), 7.19 (d, J = 7.6Hz, 1H), 7.07-6.98 (m, 2H), 4.75-4.68 (m, 1H), 4.50-4.42 (m, 2H), 4.37-4.25 (m, 2H), 3.92 (s, 3H), 3.82 (t, 2H), 3.76 -3.50 (m, 9H), 2.18 (bs, 1H), 1.65 (d, J = 5.2Hz, 3H), 1.36-1.22 (m, 1H), 0.95 (S, 9H) (see FIG. 2).
MS: C ₃₉ H ₄₅ Cl ₂ FN ₃ O ₁₀ ([M + H] ⁺ ) Calculated value: 804; Measured value: 804.2.

ラセミ−４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキソスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸（３０６ｍｇ、０．５ｍｍｏｌ、実施例８の合成におけるステップ１を参照）と１−クロロエチル（２−（２−メトキシエトキシエトキシ）エチル）カーボネート（６０９ｍｇ、２．２５ｍｍｏｌ）のジメチルホルムアミド（１０ｍｌ）溶液にＮａＯＨ（３ｍｍｏｌ、９７５ｍｇ、６ｅｑ）を添加し、窒素ガス雰囲気中で終夜攪拌し、その後、１０ｍｌの水を添加し、この混合物を酢酸エチル（ＥＡ）で抽出し（２×１５ｍｌ）、有機層を合わせ、水で洗浄し、さらに無水Ｎａ_２ＳＯ_４により乾燥し、溶剤を真空下で除去し、得られた残留物をシリカゲルカラムクロマトグラフィーにより精製し、白色の固体として目的化合物（１００ｍｇ、収率２４％）を得た。
^１Ｈ ＮＭＲ（ＣＤＣｌ_３、４００ＭＨｚ）：δ１０．６２（ｓ，１Ｈ），８．５０（ｄ，Ｊ＝４．２Ｈｚ，１Ｈ），７．７９（ｄ，Ｊ＝０．６Ｈｚ，１Ｈ），７．７４（ｄ，Ｊ＝４．２Ｈｚ，１Ｈ），７．６１（ｓ，１Ｈ），７．４７（ｔ，Ｊ＝６．４Ｈｚ，１ Ｈ），７．３３（ｄ，Ｊ＝７．６Ｈｚ，１Ｈ），７．１０−７．２５（ｍ，２Ｈ），７．０２（ｔ，Ｊ＝７．８Ｈｚ，１Ｈ），４．７０（ｄ，Ｊ＝４．８Ｈｚ，１Ｈ），４．４０−４．５０（ｍ，３Ｈ），３．９３（ｓ，３Ｈ），３．７８−３．８０（ｍ，２Ｈ）， ３．７２（ｄ，Ｊ＝９．０Ｈｚ，４Ｈ），３．６４（ｓ，９Ｈ），３．５０−３．５４（ｍ，２Ｈ），３．３６（ｓ，３Ｈ），１．２３−１．３５（ｍ，１Ｈ），０．９５（ｓ， ９Ｈ），０．９０（ｓ，１Ｈ）；
ＭＳ：Ｃ_４１Ｈ_４９Ｃｌ_２ＦＮ_３Ｏ_１１（［Ｍ＋Ｈ］^＋）計算値：８４８；実測値：８４８．３。 Example 9: Racemic-12-oxo-2,5,8,11,13-pentaoxapentadecane-14-yl-4-((2'S, 3R, 4'S, 5'R) -6-chloro -4'-(3-Chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidine] -5'-carboxamide) -3-methoxybenzoic acid ester (racemic-) Synthesis of SIP-PEG-3)

Racem-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [ Dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoic acid (306 mg, 0.5 mmol, see step 1 in the synthesis of Example 8) and 1-chloroethyl (2- (2- (2- (2- (2-) NaOH (3 mmol, 975 mg, 6 eq) was added to a solution of methoxyethoxyethoxy) ethyl) carbonate (609 mg, 2.25 mmol) in dimethylformamide (10 ml), stirred overnight in a nitrogen gas atmosphere, and then 10 ml of water was added. The mixture was extracted with ethyl acetate (EA) (2 x 15 ml), the organic layers were combined, washed with water, dried over anhydrous Na ₂ SO ₄ , and the solvent was removed under vacuum to give. The residue was purified by silica gel column chromatography to obtain the target compound (100 mg, yield 24%) as a white solid.
^{1 1} H NMR (CDCl ₃ , 400 MHz): δ10.62 (s, 1H), 8.50 (d, J = 4.2 Hz, 1H), 7.79 (d, J = 0.6 Hz, 1H), 7 .74 (d, J = 4.2Hz, 1H), 7.61 (s, 1H), 7.47 (t, J = 6.4Hz, 1H), 7.33 (d, J = 7.6Hz) , 1H), 7.10-7.25 (m, 2H), 7.02 (t, J = 7.8Hz, 1H), 4.70 (d, J = 4.8Hz, 1H), 4.40 -4.50 (m, 3H), 3.93 (s, 3H), 3.78-3.80 (m, 2H), 3.72 (d, J = 9.0Hz, 4H), 3.64 (S, 9H), 3.50-3.54 (m, 2H), 3.36 (s, 3H), 1.23-1.35 (m, 1H), 0.95 (s, 9H), 0.90 (s, 1H);
MS: C ₄₁ H ₄₉ Cl ₂ FN ₃ O ₁₁ ([M + H] ⁺ ) Calculated value: 848; Measured value: 848.3.

ラセミ−４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキソスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸（４．０ｇ、６．５ｍｍｏｌ、実施例８の合成にかけるステップ１を参照）と（Ｒ）−Ｎ，Ｎ−ジメチル−１−フェニル−１−エチルアミン（１．１２ｇ、７．４９ｍｍｏｌ、（Ｒ）−Ｎ，Ｎ−ｄｉｍｅｔｈｙｌ−１−ｐｈｅｎｙｌｅｔｈａｎ−１−ａｍｉｎｅ）との混合物を酢酸エチルに分散させ、６０℃に加熱してこの温度で２時間撹拌し、その後、常温まで冷却して終夜攪拌した。析出した固体を濾過して低温酢酸エチルで洗浄し、乾燥後、白色の固体としてＲ−アミン塩４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキソスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸−（Ｒ）−Ｎ，Ｎ−ジメチル−１−フェニル−１−エチルアミン塩（２．０ｇ）を得た。
^１Ｈ ＮＭＲ（ｄ_６−ＤＭＳＯ，４００ＭＨｚ）：δ１０．７５（ｓ，１Ｈ），１０．４７（ｓ，１Ｈ），８．３８（ｄ，Ｊ＝８．４Ｈｚ，１Ｈ），７．７０（ｄ，Ｊ＝８．０Ｈｚ，１Ｈ），７．６１（ｔ，Ｊ＝７．０Ｈｚ，１Ｈ），７．５５−７．５８（ｍ，２Ｈ），７．３７（ｔ，Ｊ＝７．４Ｈｚ，１Ｈ），７．２５−７．３３（ｍ，４Ｈ），７．２２（ｔ，Ｊ＝６．８Ｈｚ，１Ｈ），７．１７（ｔ，Ｊ＝８．０Ｈｚ，１Ｈ），７．００（ｄ，Ｊ＝８．４Ｈｚ，１Ｈ），６．６７（ｓ，１Ｈ），４．６７（ｔ，Ｊ＝７．６Ｈｚ，１Ｈ），４．４７（ｄ，Ｊ＝９．６Ｈｚ，１Ｈ），３．９０（ｓ，３Ｈ），３．６５−３．９０（ｍ，１Ｈ），３．２０−３．４０（ｍ，２Ｈ），２．０８（ｓ，６Ｈ），１．２９−１．３２（ｍ，１Ｈ），１．２６（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ），０．９１ （ｓ，９Ｈ），０．７６（ｄ，Ｊ＝１４Ｈｚ，１Ｈ）（図３を参照）。 Example 10: A compound represented by the following structural formula (4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluoro) Phenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidine] -5'-carboxamide) -3-methoxybenzoic acid- (R) -N, N-dimethyl-1-phenyl-1 -Synthesis of ethylamine salt, R-amine salt)

Racem-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [ Dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoic acid (4.0 g, 6.5 mmol, see step 1 for synthesis in Example 8) and (R) -N, A mixture with N-dimethyl-1-phenyl-1-ethylamine (1.12 g, 7.49 mmol, (R) -N, N-dimethyl-1-phenyllethan-1-amine) was dispersed in ethyl acetate and heated to 60 ° C. The mixture was heated to the above temperature and stirred at this temperature for 2 hours, then cooled to room temperature and stirred overnight. The precipitated solid is filtered, washed with low-temperature ethyl acetate, dried, and then converted into a white solid with R-amine salt 4-((2'S, 3'R, 4'S, 5'R) -6-chloro-. 4'-(3-Chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidine] -5'-carboxamide) -3-methoxybenzoic acid- (R)- N, N-dimethyl-1-phenyl-1-ethylamine salt (2.0 g) was obtained.
¹ H NMR (d _6- DMSO, 400 MHz): δ10.75 (s, 1H), 10.47 (s, 1H), 8.38 (d, J = 8.4 Hz, 1H), 7.70 (d) , J = 8.0Hz, 1H), 7.61 (t, J = 7.0Hz, 1H), 7.55-7.58 (m, 2H), 7.37 (t, J = 7.4Hz, 1H), 7.25-7.33 (m, 4H), 7.22 (t, J = 6.8Hz, 1H), 7.17 (t, J = 8.0Hz, 1H), 7.00 ( d, J = 8.4Hz, 1H), 6.67 (s, 1H), 4.67 (t, J = 7.6Hz, 1H), 4.47 (d, J = 9.6Hz, 1H), 3.90 (s, 3H), 3.65-3.90 (m, 1H), 3.20-3.40 (m, 2H), 2.08 (s, 6H), 1.29-1. 32 (m, 1H), 1.26 (d, J = 6.8Hz, 3H), 0.91 (s, 9H), 0.76 (d, J = 14Hz, 1H) (see FIG. 3).

４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキソスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸−（Ｒ）−Ｎ，Ｎ−ジメチル−１−フェニル−１−エチルアミン塩（３８２ｍｇ、０．５ｍｍｏｌ）と１−クロロエチル（２−（２−メトキシエトキシエトキシ）エチル）カーボネート（６０９ｍｇ、２．２５ｍｍｏｌ）のジメチルホルムアミド（１０ｍｌ）溶液に、ＮａＯＨ（３ｍｍｏｌ、９７５ｍｇ、６ｅｑ）を添加し、窒素ガス雰囲気中で終夜攪拌し、その後、１０ｍｌの水を添加し、この混合物を酢酸エチルで抽出し（２×１５ｍｌ）、有機層を合わせ、水で洗浄し、さらに無水Ｎａ_２ＳＯ_４により乾燥し、溶剤を真空下で除去し、得られた残留物をシリカゲルカラムクロマトグラフィーにより精製し、白色の固体として目的化合物（９８ｍｇ、収率２３％）を得た。［α］^２０ _Ｄ＝−１３８^ｏ（０．１ｇ／１００ｍｌ、ＣＨ_３ＯＨ）。
^１Ｈ ＮＭＲ（ＣＤＣｌ_３、４００ＭＨｚ）：δ１０．６２（ｓ，１Ｈ），８．５１（ｄ，Ｊ＝８．８Ｈｚ，１Ｈ），７．９２（ｓ，１Ｈ），７．７３（ｄ，Ｊ＝８．４Ｈｚ，１Ｈ），７．６０（ｓ，１Ｈ），７．４７（ｔ，Ｊ＝６．８Ｈｚ，１Ｈ），７．３３（ｄ，Ｊ＝７．６Ｈｚ，１Ｈ），７．１０−７．２５（ｍ，２Ｈ），７．０２（ｔ，Ｊ＝７．８Ｈｚ，１Ｈ），４．６８−４．８０（ｍ，１Ｈ），４．４０−４．５０（ｍ，３Ｈ），３．９３（ｓ，３Ｈ），３．８０−３．８８（ｍ，２Ｈ），３．７０−３．８０（ｍ，４Ｈ），３．６０−３．７０（ｍ，９Ｈ），３．５０−３．５８（ｍ，２Ｈ），３．３６（ｓ，３Ｈ），１．２３−１．３５（ｍ，１Ｈ），０．９５（ｓ，９Ｈ），０．８５−０．９０（ｍ，１Ｈ）（図４を参照）；
ＭＳ：Ｃ_４１Ｈ_４９Ｃｌ_２ＦＮ_３Ｏ_１１（［Ｍ＋Ｈ］^＋）計算値：８４８；実測値：８４８．３。 Example 11: Chiral-12-oxo-2,5,8,11,13-pentaoxapentadecane-14-yl-4-((2'S, 3R, 4'S, 5'R) -6-chloro -4'-(3-Chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoic acid ester (chiral-) Synthesis of R-SIP-PEG-3)

4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole] -3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoic acid- (R) -N, N-dimethyl-1-phenyl-1-ethylamine salt (382 mg, 0.5 mmol) and 1-chloroethyl NaOH (3 mmol, 975 mg, 6 eq) was added to a solution of (2- (2-methoxyethoxyethoxy) ethyl) carbonate (609 mg, 2.25 mmol) in dimethylformamide (10 ml), and the mixture was stirred overnight in a nitrogen gas atmosphere. Then 10 ml of water was added, the mixture was extracted with ethyl acetate (2 x 15 ml), the organic layers were combined, washed with water and further dried with anhydrous Na ₂ SO ₄ to remove the solvent under vacuum. The obtained residue was purified by silica gel column chromatography to obtain the target compound (98 mg, 23% yield) as a white solid. [Α] ²⁰ _D = -138 ^o (0.1 g / 100 ml, CH ₃ OH).
^{1 1} H NMR (CDCl ₃ , 400 MHz): δ10.62 (s, 1H), 8.51 (d, J = 8.8 Hz, 1H), 7.92 (s, 1H), 7.73 (d, J) = 8.4Hz, 1H), 7.60 (s, 1H), 7.47 (t, J = 6.8Hz, 1H), 7.33 (d, J = 7.6Hz, 1H), 7.10 -7.25 (m, 2H), 7.02 (t, J = 7.8Hz, 1H), 4.68-4.80 (m, 1H), 4.40-4.50 (m, 3H) , 3.93 (s, 3H), 3.80-3.88 (m, 2H), 3.70-3.80 (m, 4H), 3.60-3.70 (m, 9H), 3 .50-3.58 (m, 2H), 3.36 (s, 3H), 1.23-1.35 (m, 1H), 0.95 (s, 9H), 0.85-0.90 (M, 1H) (see FIG. 4);
MS: C ₄₁ H ₄₉ Cl ₂ FN ₃ O ₁₁ ([M + H] ⁺ ) Calculated value: 848; Measured value: 848.3.

ラセミ−４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキソスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸（３０６ｍｇ、０．５ｍｍｏｌ）と１−クロロエチル（２，５，８，１１，１４，１７，２０−ヘプタオキサドコサン−２２−イル）カーボネート（１−ｃｈｌｏｒｏｅｔｈｙｌ（２，５，８，１１，１４，１７，２０−ｈｅｐｔａｏｘａｄｏｃｏｓａｎ−２２−ｙｌ）ｃａｒｂｏｎａｔｅ）（１．００ｇ、２．２５ｍｍｏｌ）のジメチルホルムアミド（１０ｍｌ）溶液に、ＮａＯＨ（３ｍｍｏｌ、９７５ｍｇ、６ｅｑ）を添加し、窒素ガス雰囲気中で終夜攪拌し、その後、１０ｍｌの水を添加し、この混合物を酢酸エチルで抽出し（２×１５ｍｌ）、有機層を合わせ、水で洗浄し、さらに無水Ｎａ_２ＳＯ_４により乾燥し、溶剤を真空下で除去し、得られた残留物をシリカゲルカラムクロマトグラフィーにより精製し、目的化合物（７７ ｍｇ、収率１５％）を得た。
計算分子式（Ｍ＋Ｈ）：Ｃ_４９Ｈ_６５Ｃｌ_２ＦＮ_３Ｏ_１５；計算分子量：１０２４．３７７７；測定値ＨＲＭＳ（Ｃ_４９Ｈ_６５Ｃｌ_２ＦＮ_３Ｏ_１５）：１０２４．３７７２。 Example 12: Racemic-24-oxo-2,5,8,11,14,17,20,23,25-Nonaoxaheptacosan-26-yl-4-((2'S, 3R, 4'S) , 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidine] -5'-carboxamide)- Synthesis of 3-methoxybenzoic acid ester (racemic-SIP-PEG-7)

Racem-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [ Dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoic acid (306 mg, 0.5 mmol) and 1-chloroethyl (2,5,8,11,14,17,20-heptaoxa) Docosan-22-yl) carbonate (1-chromoethyl (2,5,8,11,14,17,20-heptaoxadocosan-22-yl) solvent) (1.00 g, 2.25 mmol) dimethylformamide (10 ml) To the solution, NaOH (3 mmol, 975 mg, 6 eq) was added and stirred overnight in a nitrogen gas atmosphere, then 10 ml of water was added and the mixture was extracted with ethyl acetate (2 x 15 ml) to remove the organic layer. The mixture was combined, washed with water, further dried with anhydrous Na ₂ SO ₄ , the solvent was removed under vacuum, and the obtained residue was purified by silica gel column chromatography to obtain the target compound (77 mg, yield 15%). Got
Calculated molecular formula (M + H): C ₄₉ H ₆₅ Cl ₂ FN ₃ O ₁₅ ; Calculated molecular weight: 1024.3777; Measured value HRMS (C ₄₉ H ₆₅ Cl ₂ FN ₃ O ₁₅ ): 1024.3772.

メトキシポリエチレングリコール１０００［ＭＰＥＧ−１０００（ＣＡＳ：９００４−７４−４、平均分子量Ｍｎ＝１０００、ＴＣＩ）、１．０ｇ、１．０ｍｍｏｌ］及びトリエチルアミン（５０５ｍｇ、５．０ｍｍｏｌ）を塩化メチレン（１０ｍｌ）に溶解し、氷浴条件でこの溶液に１−クロロエチルカルボノクロリダート（７１５ｍｇ、５．０ｍｍｏｌ）を滴下し、生成した混合物を２４時間攪拌反応させ、水洗、抽出、乾燥後、１−クロロエチルメトキシポリエチレングリコール１０００カーボネートを得て、そのまま次の反応に使用した。
ラセミ−４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキソスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸（３０６ｍｇ、０．５ｍｍｏｌ）と１−クロロエチルメトキシポリエチレングリコール１０００カーボネート（２．５３ｇ、２．２５ｍｍｏｌ）のジメチルホルムアミド（１０ｍｌ）溶液に、ＮａＯＨ（３ｍｍｏｌ、９７５ｍｇ、６ｅｑ）を添加し、窒素ガス雰囲気中で終夜攪拌し、その後、１０ｍｌの水を添加し、この混合物を酢酸エチルで抽出し（２×１５ｍｌ）、有機層を合わせ、水で洗浄し、さらに無水Ｎａ_２ＳＯ_４により乾燥し、溶剤を真空下で除去し、得られた残留物を分取液体クロマトグラフィーにより精製し、目的化合物（１２８ｍｇ、収率１５％）を得た。ＭＡＬＤＩ−ＴＯＦ−ＭＳ：約１７００（図５を参照）。 Example 13: Racemic-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl- Synthesis of 2-oxospiro [dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoate methoxypolyethylene glycol 1000 carbonate (racemic-SIP-MPEG-1000)

Methylene chloride (10 ml) with methoxypolyethylene glycol 1000 [MPEG-1000 (CAS: 9004-74-4, average molecular weight Mn = 1000, TCI), 1.0 g, 1.0 mmol] and triethylamine (505 mg, 5.0 mmol) After dissolution, 1-chloroethyl carbonochloridate (715 mg, 5.0 mmol) was added dropwise to this solution under ice bath conditions, and the resulting mixture was stirred and reacted for 24 hours, washed with water, extracted, dried, and then 1-chloroethyl. A methoxypolyethylene glycol 1000 carbonate was obtained and used as it was in the next reaction.
Racem-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [ Dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoic acid (306 mg, 0.5 mmol) and 1-chloroethylmethoxypolyethylene glycol 1000 carbonate (2.53 g, 2.25 mmol) dimethyl To a solution of formamide (10 ml), NaOH (3 mmol, 975 mg, 6 eq) was added and stirred overnight in a nitrogen gas atmosphere, then 10 ml of water was added and the mixture was extracted with ethyl acetate (2 x 15 ml). , The organic layers are combined, washed with water, further dried with anhydrous Na ₂ SO ₄ , the solvent is removed under vacuum and the resulting residue is purified by preparative liquid chromatography to obtain the compound of interest (128 mg, yield). Rate 15%) was obtained. MALDI-TOF-MS: Approximately 1700 (see FIG. 5).

１−クロロエチルメトキシポリエチレングリコール２０００カーボネートは、実施例１３にかける１−クロロエチルメトキシポリエチレングリコール１０００カーボネートの合成方法を参照して原料であるメトキシポリエチレングリコール２０００（ＣＡＳ：９００４−７４−４、平均分子量Ｍｎ＝２０００、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ）から調製することができる。
ラセミ−４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキサスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸（３０６ｍｇ、０．５ｍｍｏｌ）と１−クロロエチルメトキシポリエチレングリコール２０００カーボネート（４．８０ｇ、２．２５ｍｍｏｌ）のジメチルホルムアミド（１０ｍｌ）溶液に、ＮａＯＨ（３ｍｍｏｌ、９７５ｍｇ、６ｅｑ）を添加し、窒素ガス雰囲気中で終夜攪拌し、その後、１０ｍｌの水を添加し、この混合物を酢酸エチルで抽出し（２×１５ｍｌ）、有機層を合わせ、水で洗浄し、さらに無水Ｎａ_２ＳＯ_４により乾燥し、溶剤を真空下で除去し、得られた残留物を分取液体クロマトグラフィーにより精製し、目的化合物（２２０ｍｇ、収率１７％）を得た。ＭＡＬＤＩ−ＴＯＦ−ＭＳ：約２６００（図６を参照）。 Example 14: Racemic-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl- Synthesis of 2-oxospiro [dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoate methoxypolyethylene glycol 2000 carbonate (racemic-SIP-MPEG-2000)

1-Chloroethylmethoxypolyethylene glycol 2000 carbonate is a raw material of methoxypolyethylene glycol 2000 (CAS: 9004-74-4, average molecular weight) with reference to the method for synthesizing 1-chloroethylmethoxypolyethylene glycol 1000 carbonate applied to Example 13. It can be prepared from Mn = 2000, Sigma-Aldrich).
Racem-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxaspiro [ Dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoic acid (306 mg, 0.5 mmol) and 1-chloroethylmethoxypolyethylene glycol 2000 carbonate (4.80 g, 2.25 mmol) dimethyl To a solution of formamide (10 ml), NaOH (3 mmol, 975 mg, 6 eq) was added and stirred overnight in a nitrogen gas atmosphere, then 10 ml of water was added and the mixture was extracted with ethyl acetate (2 x 15 ml). , The organic layers are combined, washed with water, further dried with anhydrous Na ₂ SO ₄ , the solvent is removed under vacuum and the resulting residue is purified by preparative liquid chromatography to obtain the desired compound (220 mg, yield). The rate was 17%). MALDI-TOF-MS: Approximately 2600 (see FIG. 6).

質量分析計による計算値（Ｍ＋Ｈ）^＋：７９０
質量分析計による測定値（Ｍ＋Ｈ）^＋：７９０．２
実施例１６：

質量分析計による計算値（Ｍ＋Ｈ）^＋：８１８
質量分析計による測定値（Ｍ＋Ｈ）^＋：８１８．３
実施例１７：

質量分析計による計算値（Ｍ＋Ｈ）^＋：８６０
質量分析計による測定値（Ｍ＋Ｈ）^＋：８６０．４ Example 15-17: Synthesis of racemic compound represented by the following structural formula (see Example 8 for the synthetic route)
Example 15:

Calculated by mass spectrometer (M + H) ⁺ : 790
Measured value by mass spectrometer (M + H) ⁺ : 790.2
Example 16:

Calculated by mass spectrometer (M + H) ⁺ : 818
Measured value by mass spectrometer (M + H) ⁺ : 818.3
Example 17:

Calculated by mass spectrometer (M + H) ⁺ : 860
Measured value by mass spectrometer (M + H) ⁺ : 860.4

質量分析計による計算値（Ｍ＋Ｈ）^＋：７９０
質量分析計による測定値（Ｍ＋Ｈ）^＋：７９０．３
実施例１９：

質量分析計による計算値（Ｍ＋Ｈ）^＋：８０４
質量分析計による測定値（Ｍ＋Ｈ）^＋：８０４．３
実施例２０：

質量分析計による計算値（Ｍ＋Ｈ）^＋：１００６
質量分析計による測定値（Ｍ＋Ｈ）^＋：１００６．４ Examples 18 to 20: Synthesis of racemic compounds represented by the following structural formulas (see Examples 9 and 12 for synthetic pathways)
Example 18:

Calculated by mass spectrometer (M + H) ⁺ : 790
Measured value by mass spectrometer (M + H) ⁺ : 790.3
Example 19:

Calculated value by mass spectrometer (M + H) ⁺ : 804
Measured value by mass spectrometer (M + H) ⁺ : 804.3
Example 20:

Calculated by mass spectrometer (M + H) ⁺ : 1006
Measured value by mass spectrometer (M + H) ⁺ : 1006.4

クロマトグラフィー条件：Ｉｉｎｅｒｔｓｉｌ ＯＤＳ−３ カラム（４．６ｍｍ×１５０ｍｍ、５μｍ）；移動相：アセトニトリル−２０％リン酸溶液（４０：６０、Ｖ／Ｖ）；流速：１ｍｌ／ｍｉｎ；検出波長：２８６ｎｍ；注入量：２０μｌ；温度：室温。このクロマトグラフィー条件下で、濃度が適切なラセミ−ＳＩＰ標準品溶液を注入して測定したところ、ラセミ−ＳＩＰピークの保持時間が４．５ｍｉｎであり、その不純物が主ピークの測定に干渉しなかった。
ラセミ−ＳＩＰ標準品の母液を正確に秤量し、１０ｍｌのメスフラスコに入れ、液面を目盛りに合わせるようにアセトニトリルを加えて希釈し、良く振って濃度がそれぞれ０．１、０．５、１．０、２．０及び３．０μｇ／ｍｌの一連の標準溶液を得た。前記クロマトグラフィー条件で検出し、ピーク面積を記録し、ピーク面積（Ｙ）を濃度（Ｘ）に対してプロットし、回帰分析し、回帰方程式（Ｙ＝８８．８５Ｘ＋０．２２２１、ｒ^２＝０．９９９８）を得て、この式に基づいてサンプルの濃度を計算した。
過剰なラセミ−ＳＩＰ、ラセミ−４−（（２’Ｓ，３’Ｒ，４’Ｓ，５’Ｒ）−６−クロロ−４’−（３−クロロ−２−フルオロフェニル）−２’−ネオペンチル−２−オキソスピロ［ジヒドロインドール−３，３’−ピロリジン］−５’−カルボキサミド）−３−メトキシ安息香酸（ラセミ−ＳＩＰ−酸）（実施例８における反応を参照すると、ラセミ−ＳＩＰ−ＰＥＧ−２の前駆体である）、ラセミ−ＳＩＰ−ＰＥＧ−２（即ち、実施例８の化合物）、ラセミ−ＳＩＰ−ＰＥＧ−３（即ち、実施例９の化合物）、キラル−Ｒ−ＳＩＰ−ＰＥＧ−３（即ち、実施例１１の化合物）、ラセミ−ＳＩＰ−ＰＥＧ−７（即ち、実施例１２の化合物）、ラセミ−ＳＩＰ−ＭＰＥＧ−１０００（即ち、実施例１３的化合物）、ラセミ−ＳＩＰ−ＭＰＥＧ−２０００（即ち、実施例１４の化合物）をそれぞれ栓付試験管に入れ、適量の水を加え、溶解が進行しなくなるまで超音波処理し、振盪器に入れ（１００ ｒ／ｍｉｎ）、（２５±１）℃で２４ｈ振盪し、溶液が平衡に到達した後、各化合物の飽和溶液を取り出し、遠心機に置いて、１００００ ｒ／ｍｉｎで１０ｍｉｎ遠心し、上澄み液を正確に吸い取り、アセトニトリルで定量的に希釈してから、サンプルを注入して測定し、ＳＩＰ検量線から異なる誘導体の水への溶解度を近似的に計算した。
３、実験結果
溶解度試験の結果から、ラセミ−ＳＩＰ及びラセミ−ＳＩＰ−酸は、水への溶解度が極めて低く（＜０．１ｍｇ／ｍｌ）、ほとんど溶解しないが、ラセミ−ＳＩＰ−ＰＥＧ−２（即ち、実施例８の化合物）の溶解度＞０．４ｍｇ／ｍｌで、ラセミ−ＳＩＰ−ＰＥＧ−３（即ち、実施例９の化合物）の溶解度＞３ｍｇ／ｍｌで、キラル−Ｒ−ＳＩＰ−ＰＥＧ−３（即ち、実施例１１の化合物）の溶解度＞８ｍｇ／ｍｌで、ラセミ−ＳＩＰ−ＰＥＧ−７（即ち、実施例１２の化合物）の溶解度が３０ｍｇ／ｍｌで、ラセミ−ＳＩＰ−ＭＰＥＧ−１０００（即ち、実施例１３的化合物）の溶解度＞１００ｍｇ／ｍｌで、ラセミ−ＳＩＰ−ＭＰＥＧ−２０００（即ち、実施例１４の化合物）の溶解度＞１００ｍｇ／ｍｌであることが示された。ＰＥＧ（即ち、−ＣＨ_２−ＣＨ_２−Ｏ−）鎖長が増加するにつれて、溶解性がより良くなるとの結論が得られた。 Test Examples Improvement effect on water solubility of the compound of the present invention 1. Experimental purpose The improvement effect on water solubility of the spiroindolone-based compound by the polyethylene glycol carbonate structure of the compound of the present invention is investigated.
2. Experimental method Weigh accurately 30.00 mg of the standard racemic-SIP (racemic-spirolinone pyrolidine carboxamide; Org. Acetonitrile Res. Dev. 2013; 17 (2): 247-256) into a 10 ml volumetric flask. It was added, and acetonitrile was added to dissolve it, and the liquid level was adjusted to the scale to prepare a racemic-SIP mother liquor of 3.00 mg / ml, which was stored at low temperature and in the dark.

Chromatographic conditions: Room temperature ODS-3 column (4.6 mm × 150 mm, 5 μm); Mobile phase: Acetonitrile-20% phosphate solution (40:60, V / V); Flow velocity: 1 ml / min; Detection wavelength: 286 nm; Injection volume: 20 μl; Temperature: Room temperature. When measured by injecting a racemic-SIP standard solution having an appropriate concentration under these chromatographic conditions, the retention time of the racemic-SIP peak was 4.5 min, and the impurities did not interfere with the measurement of the main peak. It was.
Accurately weigh the mother liquor of the Lasemi-SIP standard product, put it in a 10 ml volumetric flask, add acetonitrile to dilute it so that the liquid level is aligned with the scale, and shake well to make the concentrations 0.1, 0.5, 1 respectively. A series of standard solutions of 0.0, 2.0 and 3.0 μg / ml were obtained. Detected under the above chromatographic conditions, the peak area is recorded, the peak area (Y) is plotted against the concentration (X), regression analysis is performed, and the regression equation (Y = 88.85X + 0.2221, r ² = 0. 9998) was obtained, and the concentration of the sample was calculated based on this equation.
Excess Racemic-SIP, Racemic-4-((2'S, 3'R, 4'S, 5'R) -6-Chloro-4'-(3-Chloro-2-fluorophenyl) -2'- Neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidin] -5'-carboxamide) -3-methoxybenzoic acid (racemic-SIP-acid) (referring to the reaction in Example 8) racemic-SIP-PEG -2 precursor), racemic-SIP-PEG-2 (ie, compound of Example 8), racemic-SIP-PEG-3 (ie, compound of Example 9), chiral-R-SIP-PEG -3 (ie, the compound of Example 11), Racemic-SIP-PEG-7 (ie, the compound of Example 12), Racemic-SIP-PEG-1000 (ie, the compound of Example 13), Racemic-SIP- Each of MPEG-2000 (that is, the compound of Example 14) was placed in a stoppered test tube, an appropriate amount of water was added, ultrasonic treatment was performed until dissolution did not proceed, and the mixture was placed in a shaker (100 r / min). Shake at 25 ± 1) ° C. for 24 hours, after the solution reaches equilibrium, remove the saturated solution of each compound, place in a centrifuge, centrifuge at 10000 r / min for 10 min, accurately absorb the supernatant and with acetonitrile. After quantitatively diluting, the sample was injected and measured, and the solubility of different derivatives in water was approximately calculated from the SIP calibration line.
3. Experimental results From the results of the solubility test, racemic-SIP and racemic-SIP-acid have extremely low solubility in water (<0.1 mg / ml) and are hardly soluble, but racemic-SIP-PEG-2 ( That is, the solubility of (compound of Example 8)> 0.4 mg / ml and the solubility of racemic-SIP-PEG-3 (ie, the compound of Example 9)> 3 mg / ml, chiral-R-SIP-PEG- 3 (ie, the compound of Example 11) has a solubility of> 8 mg / ml, and Racemic-SIP-PEG-7 (ie, the compound of Example 12) has a solubility of 30 mg / ml, Racemic-SIP-PEG-1000 (ie, the compound of Example 12). That is, it was shown that the solubility of the compound of Example 13)> 100 mg / ml and the solubility of racemic-SIP-PEG-2000 (that is, the compound of Example 14)> 100 mg / ml. It was concluded that as the PEG (ie-CH _2- CH _2- O-) chain length increased, the solubility became better.

Inhibitory effect of the compound of the present invention on the growth of transplanted tumors of SJSA-1 nude mice 1. Experimental purpose Racemic-SIP as a known chiral compound, racemic-SIP-PEG-2 as a chiral compound of the present invention (that is, implementation Example 8 compound), racemic-SIP-PEG-3 (ie, Example 9 compound), chiral-R-SIP-PEG-3 (ie, Example 11 compound), racemic-SIP-PEG-7 (ie, Example 11 compound). That is, the inhibitory effect of the compound of Example 12) and racemic-SIP-MPEG-2000 (that is, the compound of Example 14) on the growth of SJSA-1 nude mouse transplanted tumor is examined.
2. Experimental animals 72 Balb / c / nu nude mice (body weight 15-19 g, female) were provided by Beijing Huafukang Biotechnology Co., Ltd. Certificate number: SCXK (K computer) 2014-0004.
3. Experimental drugs and reagents (1) Drugs Adriamycin: Positive control drug Racemic-SIP: (Org. Process Res. Dev. 2013; 17 (2): 247-256) Milky white powder;
Racem-SIP-PEG-2 (ie, compound of Example 8): white powder Racem-SIP-PEG-3 (ie, compound of Example 9): white powder Chiral-R-SIP-PEG-3 (ie, compound of Example 9) That is, the compound of Example 11): white powder racem-SIP-PEG-7 (that is, the compound of Example 12): white powder racem-SIP-PEG-2000 (that is, the compound of Example 14): white. (2) Preparation method of drug Adriamycin: Prepared to 0.3 mg / ml with sterile physiological saline, and the dose was 3.0 mg / kg and the dose volume was 10 ml / kg.
Racem-SIP: Prepared into a solution with a concentration of 1.5 mg / ml using 4% DMA, 30% PEG400, 66% (volume ratio) sterile distilled water as a solvent, and the administration dose was 30.0 mg / kg and the administration volume was 20 ml / ml. It was set to kg.
Racem-SIP-PEG-2: Polyoxyethylene castor oil: 95% ethanol: Sterilized saline (volume ratio 1.5: 1.5: 7) as a solvent with concentrations of 1.5 mg / ml and 3.0 mg / Prepared in ml of drug solution. The administration dose was 15.0 mg / kg, 30.0 mg / kg, and the administration volume was 10 ml / kg.
Racemic-SIP-PEG-3: Prepared as a drug solution having a concentration of 3.0 mg / ml with sterile physiological saline. The administration dose was 30.0 mg / kg. The administration volume was 10 ml / kg.
Chiral-R-SIP-PEG-3: Prepared as a drug solution having a concentration of 3.0 mg / ml with sterile physiological saline. The administration dose was 30.0 mg / kg. The administration volume was 10 ml / kg.
Racemic-SIP-PEG-7: Prepared as a drug solution having a concentration of 3.0 mg / ml with sterile physiological saline. The administration dose was 30.0 mg / kg. A mixture of racemic-SIP-PEG-2000: sterile physiological saline having an administration volume of 10 ml / kg was prepared to a concentration of 5.0 mg / ml. The dose was 50.0 mg / kg. The administration volume was 10 ml / kg.
(3) Administration dose and route:
The dose of adriamycin was 3.0 mg / kg, and it was administered by intraperitoneal injection once every other day for a total of 6 times.
The dose of racemic-SIP was 30.0 mg / kg, and the mixture was intragastrically administered once a day, 6 times a week, for a total of 9 times.
The dose of racemic-SIP-PEG-2 was 30.0 mg / kg, and it was administered by intraperitoneal injection once a day, 6 times a week, for a total of 9 times.
The dose of racemic-SIP-PEG-3 was 30.0 mg / kg, and it was administered by intraperitoneal injection once a day, 6 times a week, for a total of 9 times.
The dose of chiral-R-SIP-PEG-3 was 30.0 mg / kg, and it was administered by intraperitoneal injection once a day, 6 times a week, for a total of 9 times.
Racemic-SIP-PEG-7: The dose was 30.0 mg / kg, and the mixture was administered by intraperitoneal injection once a day, 6 times a week, for a total of 9 times.
Racemic-SIP-mPEG-2000: The dose was 50.0 mg / kg, and the mixture was administered by intraperitoneal injection once a day, 6 times a week, for a total of 9 times.
(4) SJSA-1 cells: purchased from Shanghai Revival Biotechnology Co., Ltd. (5) Other reagents: Polyoxyethylene castor oil (Aladdin Co., Ltd .; CAS: 61791-12-6).
4. Experimental procedure Tumor cells (SJSA-1) were collected under aseptic conditions, ^{adjusted to a cell density of 1 × 10 7} cells / ml with sterile physiological saline, and subcutaneously placed on the side of a nude mouse. When 2 ml was inoculated and the tumor grew to a size of 1 cm in diameter, it was excised under aseptic conditions, cut into tumor masses having a size of 2.0 mm × 2.0 mm, and uniformly inoculated subcutaneously on the side of the nude mouse. .. When tumor volume is about 250 mm ^3, (8 days after inoculation) administered separately group nine mice, tumor growth is bad animals were excluded. The control group used saline as a control. Adriamycin dose 3.0 mg / kg, racemic-SIP dose 30.0 mg / kg, racemic-SIP-PEG-2, racemic-SIP-PEG-3, chiral-R-SIP-PEG-3 and The racemic-SIP-PEG-7 dose was 30.0 mg / kg, respectively, and the racemic-SIP-PEG-2000 dose was 50.0 mg / kg. Weight were weighed three times a week, and the length a and width b of the tumors were measured with calipers, the tumor volume was calculated by the equation ^{V = a × b 2/2} , where, a is a tumor length And b is the width of the tumor. On the 21st day after inoculation, nude mice were sacrificed by cervical spine dislocation and photographed, and then the tumor was exfoliated and weighed and photographed. Tumor RTV (relative tumor volume) and inhibition rate were calculated. At the same time, peripheral blood of nude mice was collected and blood cell count was performed.
5. Data processing The data was processed by EXCEL software, the data was represented by the mean value ± SD, and the intergroup analysis was statistically processed by t-test.
6. Results (1) Effect of solubility on dosage form As can be seen from the method for preparing pharmaceutical products, racemic-SIP-PEG-based compounds (Example 8, Example 9, Example 11, Example 12 and Example 14). Is superior in water solubility to the PEG-free compounds racem-SIP and racem-SIP-acid, and the water solubility improves as the PEG chain length increases, and Examples 9, 11, and 12 And Example 14 is more water-soluble than Example 8, can be dissolved in physiological saline as it is, is more suitable for intravenous administration, and can avoid side effects on the gastrointestinal tract due to oral preparations.
(2) Evaluation of Tumor Inhibition Rate The compound of the present invention showed a significantly stronger inhibitory effect on tumor growth than the PEG-free compound SIP due to the inclusion of PEG. Among them, when racemic-SIP-PEG-2 is administered by injection at a dose of 30.0 mg / kg, the inhibition against tumor growth is slightly superior to that when racemic-SIP containing no PEG is orally administered. Showed action. Racemic-SIP-PEG-3, Chiral-R-PEG-3, Racemic-SIP-PEG-7 at a dose of 30.0 mg / kg and Racemic-SIP-PEG-2000 at a dose of 50.0 mg / kg When administered by, it showed a stronger inhibitory effect on tumor growth as compared with the case where racemic-SIP containing no PEG was orally administered. When the inhibition rate was calculated based on the relative tumor volume, racemic-SIP-PEG-2 was 55.36%, racemic-SIP-PEG-3 was 90.79%, chiral-R-PEG-3 was 97.9%, and racemic. -SIP-PEG-7 was 96.38% and racemic-SIP-PEG-2000 was 96.02%, but SIP was only 54.89% when administered orally. Regarding the tumor inhibition rate, racemic-SIP-PEG-2 was 50.55%, racemic-SIP-PEG-3 was 92.86%, and chiral-R-PEG-3 was 97.84%, respectively, by tumor weight. Racemic-SIP-PEG-7 was 97.06% and Racemic-SIP-PEG-2000 was 96.70%, but SIP was only 49.69% when administered orally (Table 1, Figure). 7 and 8). Each treated group weighed less than the saline control group, while the chiral-R-PEG-3 treated group weighed more than the saline control group and was statistically significant. (See Table 1 and FIG. 9). It was concluded that the spiroindron polyethylene glycol carbonate compound (SIP-PEG) was superior in its antitumor activity to the non-esterified spiroindron (SIP).
(3) Evaluation of side effects As a result of comparing the body weight of each administration group with that of the solvent control group, the body weight of racemic-SIP-PEG-2 was reduced by about 9.4%, and that of racemic-SIP-PEG-3 was reduced by about 9.4%. 7.5% loss, chiral-R-PEG-3 gained about 11%, racemic-SIP-PEG-7 lost about 6.4%, racemic-SIP-PEG-2000 lost about about 6.4%. It decreased by 5.2%, but with SIP when administered orally, it decreased by about 18% and was statistically significant (see Tables 1 and 7). Therefore, it was concluded that the toxic side effect of the spiroindron polyethylene glycol carbonate compound (SIP-PEG) was smaller than that of the non-esterified spiroindron (SIP).

Claims (8)

A spiroindolone polyethylene glycol carbonate-based compound having a structure represented by the following general formula II, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

(II)
In General Formula II, Y ₁ , Y ₂ , Y ₃ and Y ₄ are independently selected from H and halogen, respectively, R ₁ is selected from H and C1-C5 alkyl groups, and R ₂ is H, C1-C5. Selected from alkyl and C1-C5 alkoxy groups,
n is an integer from 3 to 50. The spiroindolone polyethylene glycol carbonate-based compound according to claim 1, a stereoisomer thereof, wherein Y ₁ , Y ₂ , Y ₃ and Y _{4 are independently selected from H, F and Cl, respectively.} Or its pharmaceutically acceptable salt. R ₁ is selected from H and C1-C3 alkyl group, R ₂ is characterized in that it is selected from C1-C5 alkoxy group, spiro India Ron polyethylene glycol carbonate-based compound of claim 1, a stereoisomer Or its pharmaceutically acceptable salt. The spiroindolone polyethylene glycol carbonate-based compound according to claim 1, wherein the spiroindolone polyethylene glycol carbonate-based compound is selected from the following compounds, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. ..
La Semi-12-oxo -2,5,8,11,13- penta-oxa-pentadecane-14-yl -4 - ((2'S, 3R , 4'S, 5'R) -6- chloro-4 ' -(3-Chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidine] -5'-carboxamide) -3-methoxybenzoic acid ester, 4

Racemic chiral-12-oxo-2,5,8,11,13-pentaoxapentadecane-14-yl-4-((2'S, 3R, 4'S, 5'R) -6-chloro-4 '-(3-Chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidine] -5'-carboxamide) -3-methoxybenzoic acid ester,

Chiral racem-24-oxo-2,5,8,11,14,17,20,23,25-nonaoxaheptacosan-26-yl-4-((2'S, 3R, 4'S, 5'R) ) -6-Chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidine] -5'-carboxamide) -3-methoxybenzoic acid Acid ester,

Racemic Racemic-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2- Oxospiro [dihydroindole-3,3'-pyrrolidine] -5'-carboxamide) -3-methoxybenzoate methoxypolyethylene glycol 1000 carbonate,

Racemic Racemic-4-((2'S, 3'R, 4'S, 5'R) -6-chloro-4'-(3-chloro-2-fluorophenyl) -2'-neopentyl-2- oxospiro [dihydroindole-3,3'-pyrrolidin] -5'-carboxamido) -3-methoxybenzoic acid methoxy polyethylene glycol 2000 Kabone DOO,

Racemic body

La Semi-24-oxo -2,5,8,11,14,17,20,23,25- nona oxa hept co San -26- yl -4 - ((2'S, 3R , 4'S, 5 ' R) -6-Chloro-4'-(3-chlorophenyl) -2'-neopentyl-2-oxospiro [dihydroindole-3,3'-pyrrolidine] -5'-carboxamide) -3-methoxybenzoic acid ester.

Racemic body A therapeutically effective amount of the spiroindolone polyethylene glycol carbonate-based compound according to any one of claims 1 to 4, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant. An antitumor pharmaceutical composition comprising. The antitumor pharmaceutical composition according to claim 5, wherein the composition is an injection solution, a tablet or a capsule. Use of the spiroindolone polyethylene glycol carbonate-based compound according to any one of claims 1 to 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof in the preparation of a therapeutic agent for cancer. The cancers are bladder cancer, breast cancer, colon cancer, rectal cancer, renal cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, bile &#22218; cancer, ovarian cancer, pancreatic adenocarcinoma, gastric cancer, cervix. Cancer, thyroid cancer, prostate cancer, skin cancer, acute lymphocytic leukemia, acute osteoporosis leukemia, chronic osteoporosis leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin lymphoma, non-hodgkin lymphoma, hairy cell lymphoma , Berkit lymphoma, melanoma, endometrial cancer, head and neck cancer, glioblastoma and osteosarcoma. The spiroindolone-based compound represented by the following general formula III and the compound represented by the following general formula IV are subjected to a nucleophilic substitution reaction in the presence of a base to cause the spiroindolone polyethylene glycol carbonate-based compound. The method for producing a spiroindolone polyethylene glycol carbonate-based compound according to claim 1, which comprises a step of obtaining a compound.

(III)

(IV)
Here, Y ₁ , Y ₂ , Y ₃ , Y ₄ , R ₁ , R ₂ and n are synonymous with claim 1, and R ₃ is a neopentyl group.

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